Abstract P115: Building A Structurally Sound System To Understand SR-BI Function

Abstract

The interaction of scavenger receptor BI (SR-BI) and high density lipoproteins (HDL) is the key step for the bodily removal of cholesterol. The HDL/SR-BI interaction is one of the concluding steps of the reverse cholesterol transport (RCT) pathway. In RCT, cholesterol within atherosclerotic plaques is taken up by HDL particles, which then dock on SR-BI at the surface of the liver, where cholesterol can be delivered converted into bile for excretion. Humans with mutations in the gene encoding SR-BI display elevated HDL-cholesterol levels and an increased risk of cardiovascular disease (CVD). Therefore, understanding the interaction between HDL and SR-BI is crucial to discovering ways to lower plasma cholesterol and modulate CVD risk. Due to the importance of SR-BI, the goal of these studies is to resolve a structure of functional full-length human SR-BI. We have expressed and purified full-length SR-BI using an insect cell Sf9 system. In order to assess SR-BI’s functions within this system, Sf9 cells were infected with baculovirus encoding empty vector or SR-BI, or left uninfected and plated into culture dishes. Expression of SR-BI significantly increased HDL cellular association and uptake of HDL-cholesteryl esters compared to empty vector and uninfected cells. Additionally, free cholesterol efflux was increased upon SR-BI expression. Lastly, SR-BI in Sf9 cells maintained its ability to form higher order oligomers in cells, which is crucial, as SR-BI oligomerization is important in cholesterol transport. Additionally, size exclusion chromatography shows that purified SR-BI is able to form oligomers in micelles. Lastly, using microscale thermophoresis, we demonstrated that purified SR-BI binds to its ligands (apolipoprotein A-I, holoparticle HDL, and oxidized LDL) with high affinity, regardless of the glycosylation state of SR-BI. Together, these assays represent the first steps in resolving a high-resolution structure of human full-length SR-BI and provide promise for delineating the ways in which the HDL/SR-BI relationship allows for efficient cholesterol clearance.